Sam:LabNotes/Human/2009-4-12: Difference between revisions
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>Sam Chiang |
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==Results== | ==Results== | ||
'''Primer 1-1''' | |||
{| {{table}} | {| {{table}} | ||
| | | align="center" style="background:#f0f0f0;"|'''Content''' | ||
| align="center" style="background:#f0f0f0;"|'''Description''' | |||
| align="center" style="background:#f0f0f0;"|'''Ct value''' | |||
| align="center" style="background:#f0f0f0;"|'''dCT(NTC-Sample)''' | |||
| align="center" style="background:#f0f0f0;"|'''Melting curve matched to Pos control''' | |||
|- | |- | ||
| | | | Sample||B6 (4/1) N6-No-Tre||37.91||-1.06||n | ||
|- | |- | ||
| | | | Sample||B7 (4/1) N6-No-Tre||7.72||29.13||n | ||
|- | |- | ||
| | | | Sample||D2 (4/1) N6-No-Tre||22.07||14.78||n | ||
|- | |- | ||
| | | | Sample||D4 (4/1) N6-No-Tre||6.37||30.48||n | ||
|- | |- | ||
| | | | Sample||D5 (4/1) N6-No-Tre||6.77||30.08||n | ||
|- | |- | ||
| | | | Sample||D7 (4/1) N6-No-Tre||7.37||29.48||n | ||
|- | |- | ||
| | | | Sample||D8 (4/1) N6-No-Tre||5.48||31.37||n | ||
|- | |- | ||
| | | | Sample||B3 (3/31) L4N9-Tre||36.3||0.55||y | ||
|- | |- | ||
| |- | | Sample||B4 (3/31) L4N9-Tre||33.25||3.60||y | ||
|- | |- | ||
| | | | Sample||B6 (3/31) L4N9-Tre||34.64||2.21||n | ||
|- | |- | ||
| |- | | Sample||C2 (3/31) L4N9-Tre||30.49||6.36||y | ||
|- | |- | ||
| Sample||C5 (3/31) L4N9-Tre||4.76||32.09||n | |||
|- | |- | ||
| |- | | Sample||C7 (3/31) L4N9-Tre||31.67||5.18||y | ||
|- | |- | ||
| Sample||C8 (3/31) L4N9-Tre||32.3||4.55||n | |||
|- | |- | ||
| | | | Sample||pos||24.3|||| | ||
|- | |- | ||
| | | | Blank||ntc||36.85|| | ||
|- | |- | ||
| |- | | | ||
|} | |||
'''Primer 2-1''' | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''Content''' | |||
| align="center" style="background:#f0f0f0;"|'''Description''' | |||
| align="center" style="background:#f0f0f0;"|'''Ct value''' | |||
| align="center" style="background:#f0f0f0;"|'''dCT(NTC-Sample)''' | |||
| align="center" style="background:#f0f0f0;"|'''Melting curve matched to Pos control''' | |||
|- | |||
| Sample||B6 (4/1) N6-No-Tre||37.18||-4.35||n | |||
|- | |||
| Sample||B7 (4/1) N6-No-Tre||30.78||2.05||n | |||
|- | |||
| Sample||D2 (4/1) N6-No-Tre||22.67||10.16||n | |||
|- | |||
| Sample||D4 (4/1) N6-No-Tre||29.83||3||y | |||
|- | |||
| Sample||D5 (4/1) N6-No-Tre||33||-0.17||n | |||
|- | |||
| Sample||D7 (4/1) N6-No-Tre||26.69||6.14||y | |||
|- | |||
| Sample||D8 (4/1) N6-No-Tre||34.23||-1.4||y | |||
|- | |||
| Sample||B3 (3/31) L4N9-Tre||31.56||1.27||y | |||
|- | |||
| Sample||B4 (3/31) L4N9-Tre||24.07||8.76||y | |||
|- | |||
| Sample||B6 (3/31) L4N9-Tre||N/A||N/A||y | |||
|- | |||
| Sample||C2 (3/31) L4N9-Tre||26.25||6.58||y | |||
|- | |||
| Sample||C5 (3/31) L4N9-Tre||N/A||N/A||n | |||
|- | |||
| Sample||C7 (3/31) L4N9-Tre||30.57||2.26||y | |||
|- | |||
| Sample||C8 (3/31) L4N9-Tre||37.38||-4.55||n | |||
|- | |- | ||
| Sample||pos||23.26|||| | |||
|- | |- | ||
| | | | Blank||ntc||32.83|||| | ||
|- | |- | ||
| | | | | ||
|} | |||
'''Primer 3-1''' | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''Content''' | |||
| align="center" style="background:#f0f0f0;"|'''Description''' | |||
| align="center" style="background:#f0f0f0;"|'''Ct value''' | |||
| align="center" style="background:#f0f0f0;"|'''dCT(NTC-Sample)''' | |||
| align="center" style="background:#f0f0f0;"|'''Melting curve matched to Pos control''' | |||
|- | |- | ||
| |- | | Sample||B6 (4/1) N6-No-Tre||41.68||-3.51||y | ||
|- | |- | ||
| Sample||B7 (4/1) N6-No-Tre||N/A||N/A||n | |||
|- | |- | ||
| |- | | Sample||D2 (4/1) N6-No-Tre||30.77||7.4||n | ||
|- | |- | ||
| Sample||D4 (4/1) N6-No-Tre||28.89||9.28||n | |||
|- | |- | ||
| |- | | Sample||D5 (4/1) N6-No-Tre||26.03||12.14||n | ||
|- | |- | ||
| Sample||D7 (4/1) N6-No-Tre||33.34||4.83||y | |||
|- | |- | ||
| |- | | Sample||D8 (4/1) N6-No-Tre||33.09||5.08||y | ||
|- | |- | ||
| Sample||B3 (3/31) L4N9-Tre||36.87||1.3||n | |||
|- | |- | ||
| |- | | Sample||B4 (3/31) L4N9-Tre||33.8||4.37||y | ||
|- | |||
| Sample||B6 (3/31) L4N9-Tre||N/A||N/A||y | |||
|- | |||
| Sample||C2 (3/31) L4N9-Tre||34.13||4.04||n | |||
|- | |||
| Sample||C5 (3/31) L4N9-Tre||40.4||-2.23||y | |||
|- | |||
| Sample||C7 (3/31) L4N9-Tre||28.02||10.15||n | |||
|- | |||
| Sample||C8 (3/31) L4N9-Tre||39.56||-1.39||y | |||
|- | |||
| Sample||pos||23.83|||| | |||
|- | |||
| Blank||ntc||38.17 | |||
|- | |||
| | |||
|} | |||
'''Primer 4-1''' | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''Content''' | |||
| align="center" style="background:#f0f0f0;"|'''Description''' | |||
| align="center" style="background:#f0f0f0;"|'''Ct value''' | |||
| align="center" style="background:#f0f0f0;"|'''dCT(NTC-Sample)''' | |||
| align="center" style="background:#f0f0f0;"|'''Melting curve matched to Pos control''' | |||
|- | |||
| Sample||B6 (4/1) N6-No-Tre||N/A||N/A||n | |||
|- | |||
| Sample||B7 (4/1) N6-No-Tre||N/A||N/A||n | |||
|- | |||
| Sample||D2 (4/1) N6-No-Tre||N/A||N/A||y | |||
|- | |||
| Sample||D4 (4/1) N6-No-Tre||N/A||N/A||n | |||
|- | |||
| Sample||D5 (4/1) N6-No-Tre||N/A||N/A||y | |||
|- | |||
| Sample||D7 (4/1) N6-No-Tre||N/A||N/A||n | |||
|- | |||
| Sample||D8 (4/1) N6-No-Tre||N/A||N/A||n | |||
|- | |||
| Sample||B3 (3/31) L4N9-Tre||N/A||N/A||y | |||
|- | |||
| Sample||B4 (3/31) L4N9-Tre||38.75||N/A||y | |||
|- | |||
| Sample||B6 (3/31) L4N9-Tre||N/A||N/A||n | |||
|- | |||
| Sample||C2 (3/31) L4N9-Tre||N/A||N/A||y | |||
|- | |||
| Sample||C5 (3/31) L4N9-Tre||N/A||N/A||n | |||
|- | |||
| Sample||C7 (3/31) L4N9-Tre||N/A||N/A||n | |||
|- | |||
| Sample||C8 (3/31) L4N9-Tre||N/A||N/A||n | |||
|- | |||
| Sample||pos||23.51||N/A|| | |||
|- | |||
| Blank||ntc||N/A||N/A | |||
|- | |||
| | |||
|} | |||
'''Primer 5-1''' | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''Content''' | |||
| align="center" style="background:#f0f0f0;"|'''Description''' | |||
| align="center" style="background:#f0f0f0;"|'''Ct value''' | |||
| align="center" style="background:#f0f0f0;"|'''dCT(NTC-Sample)''' | |||
| align="center" style="background:#f0f0f0;"|'''Melting curve matched to Pos control''' | |||
|- | |||
| Sample||B6 (4/1) N6-No-Tre||40.66||-8.72||n | |||
|- | |||
| Sample||B7 (4/1) N6-No-Tre||44.13||-12.19||n | |||
|- | |||
| Sample||D2 (4/1) N6-No-Tre||37.36||-5.42||n | |||
|- | |||
| Sample||D4 (4/1) N6-No-Tre||36.8||-4.86||n | |||
|- | |||
| Sample||D5 (4/1) N6-No-Tre||32.06||-0.12||y | |||
|- | |||
| Sample||D7 (4/1) N6-No-Tre||38.86||-6.92||n | |||
|- | |||
| Sample||D8 (4/1) N6-No-Tre||N/A||N/A||n | |||
|- | |||
| Sample||B3 (3/31) L4N9-Tre||40.66||-8.72||n | |||
|- | |||
| Sample||B4 (3/31) L4N9-Tre||N/A||N/A||y | |||
|- | |||
| Sample||B6 (3/31) L4N9-Tre||N/A||N/A||y | |||
|- | |||
| Sample||C2 (3/31) L4N9-Tre||36.89||-4.95||n | |||
|- | |||
| Sample||C5 (3/31) L4N9-Tre||N/A||N/A||y | |||
|- | |||
| Sample||C7 (3/31) L4N9-Tre||40.25||-8.31||n | |||
|- | |||
| Sample||C8 (3/31) L4N9-Tre||N/A||N/A||n | |||
|- | |||
| Sample||pos||24.02|||| | |||
|- | |||
| Blank||ntc||31.94 | |||
|- | |||
| | |||
|} | |||
'''Primer 6-1''' | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''Content''' | |||
| align="center" style="background:#f0f0f0;"|'''Description''' | |||
| align="center" style="background:#f0f0f0;"|'''Ct value''' | |||
| align="center" style="background:#f0f0f0;"|'''dCT(NTC-Sample)''' | |||
| align="center" style="background:#f0f0f0;"|'''Melting curve matched to Pos control''' | |||
|- | |- | ||
| Sample||B6 (4/1) N6-No-Tre||38.26||0.89||n | |||
|- | |- | ||
| |- | | Sample||B7 (4/1) N6-No-Tre||34.57||4.58||y | ||
|- | |- | ||
| Sample||D2 (4/1) N6-No-Tre||42.1||-2.95||n | |||
|- | |- | ||
| |- | | Sample||D4 (4/1) N6-No-Tre||39.49||-0.34||n | ||
|- | |- | ||
| Sample||D5 (4/1) N6-No-Tre||33.99||5.16||y | |||
|- | |- | ||
| |- | | Sample||D7 (4/1) N6-No-Tre||N/A||N/A||n | ||
|- | |- | ||
| Sample||D8 (4/1) N6-No-Tre||28.96||10.19||y | |||
|- | |- | ||
| |- | | Sample||B3 (3/31) L4N9-Tre||34.64||4.51||y | ||
|- | |- | ||
| Sample||B4 (3/31) L4N9-Tre||39.92||-0.77||y | |||
|- | |- | ||
| |- | | Sample||B6 (3/31) L4N9-Tre||35.97||3.18||y | ||
|- | |- | ||
| Sample||C2 (3/31) L4N9-Tre||36.08||3.07||y | |||
|- | |- | ||
| |- | | Sample||C5 (3/31) L4N9-Tre||N/A||N/A||y | ||
|- | |- | ||
| | | | Sample||C7 (3/31) L4N9-Tre||33.26||5.89||y | ||
|- | |- | ||
| |- | | Sample||C8 (3/31) L4N9-Tre||37.54||1.61||n | ||
|- | |- | ||
| | | | Sample||pos||26.85|||| | ||
|- | |- | ||
| | | | Blank||ntc||39.15 | ||
|- | |- | ||
| | | | ||
|} | |} |
Latest revision as of 20:42, 13 April 2009
Realtime PCR validation for L4N9 (3/31/'09) and N6 (04/01/'09) MDA amplicons[edit]
Procedure[edit]
- Experiment design
- Primers: 1-1, 2-1, 3-1, 4-1, 5-1, 6-1
- Templates: Candidates MDA amplicon. 7 samples from 3/31, 7 samples from 4/01, pos (gDNA 100pg/uL), NTC(H2O)
- Thaw gDNA(100pg/uL), Taq 2X enzyme, primers mix and diluted templates (MDA amplicon in 1/50 dilution)
- Set up realtime PCR program (decribed as following)
- Set up 12 PCR strip tubes on cool block, labed as column 1 to column 12.
- Expemriment arrangements
Column 1 -> 6 Column 7 -> 12 Primer 1-1->6-1 Primer 1-1->6-1 B6(4-01) B7(4-01) D2(4-01) D4(4-01) D5(4-01) D7(4-01) D8(4-01) B3(3-31) B4(3-31) B6(3-31) C2(3-31) C5(3-31) C7(3-31) C8(3-31) POS(100pg) NTC
- Prepare diluted primers in PCR tubes
1rxn (8+1)x2 ->20rxns Primer mix (f+r 10 uM) 1.0 20.0 uL H2O 7.0 140.0 uL ---------------------------------------- 8.0 160.0
- Transfer 8 uL diluted primer into each reaction based on the arrangements
- Transfer 2 uL diluted templates into each reaction based on arrangements
- Prepare master mix in 1.5-mL microcentrifuge tube
1rxn (12+2)x8=112 rxns Taq (2X) 9.9 1108.8 SYBR (50X) 0.1 11.2 -------------------------------- 10.0 1120.0
- Mix well the master mix by vortexing. Seperate master into 8 PCR tubes then transer 10 uL Master mix into each reaction using multichannel pipettor.
- Gently vortex and spin down the PCR reaction tubes. Place in Chromo4 machine immediately to perform realtime PCR reaction.
- Program: 94C 2min-> (94C 40sec-> 52C 40sec -> 72C sec -> plate read) x 45 -> Melting curve (52C->94C, read every 2C, hold 1min) -> 15C forever
Results[edit]
Primer 1-1
Content | Description | Ct value | dCT(NTC-Sample) | Melting curve matched to Pos control |
Sample | B6 (4/1) N6-No-Tre | 37.91 | -1.06 | n |
Sample | B7 (4/1) N6-No-Tre | 7.72 | 29.13 | n |
Sample | D2 (4/1) N6-No-Tre | 22.07 | 14.78 | n |
Sample | D4 (4/1) N6-No-Tre | 6.37 | 30.48 | n |
Sample | D5 (4/1) N6-No-Tre | 6.77 | 30.08 | n |
Sample | D7 (4/1) N6-No-Tre | 7.37 | 29.48 | n |
Sample | D8 (4/1) N6-No-Tre | 5.48 | 31.37 | n |
Sample | B3 (3/31) L4N9-Tre | 36.3 | 0.55 | y |
Sample | B4 (3/31) L4N9-Tre | 33.25 | 3.60 | y |
Sample | B6 (3/31) L4N9-Tre | 34.64 | 2.21 | n |
Sample | C2 (3/31) L4N9-Tre | 30.49 | 6.36 | y |
Sample | C5 (3/31) L4N9-Tre | 4.76 | 32.09 | n |
Sample | C7 (3/31) L4N9-Tre | 31.67 | 5.18 | y |
Sample | C8 (3/31) L4N9-Tre | 32.3 | 4.55 | n |
Sample | pos | 24.3 | ||
Blank | ntc | 36.85 | ||
Primer 2-1
Content | Description | Ct value | dCT(NTC-Sample) | Melting curve matched to Pos control |
Sample | B6 (4/1) N6-No-Tre | 37.18 | -4.35 | n |
Sample | B7 (4/1) N6-No-Tre | 30.78 | 2.05 | n |
Sample | D2 (4/1) N6-No-Tre | 22.67 | 10.16 | n |
Sample | D4 (4/1) N6-No-Tre | 29.83 | 3 | y |
Sample | D5 (4/1) N6-No-Tre | 33 | -0.17 | n |
Sample | D7 (4/1) N6-No-Tre | 26.69 | 6.14 | y |
Sample | D8 (4/1) N6-No-Tre | 34.23 | -1.4 | y |
Sample | B3 (3/31) L4N9-Tre | 31.56 | 1.27 | y |
Sample | B4 (3/31) L4N9-Tre | 24.07 | 8.76 | y |
Sample | B6 (3/31) L4N9-Tre | N/A | N/A | y |
Sample | C2 (3/31) L4N9-Tre | 26.25 | 6.58 | y |
Sample | C5 (3/31) L4N9-Tre | N/A | N/A | n |
Sample | C7 (3/31) L4N9-Tre | 30.57 | 2.26 | y |
Sample | C8 (3/31) L4N9-Tre | 37.38 | -4.55 | n |
Sample | pos | 23.26 | ||
Blank | ntc | 32.83 | ||
Primer 3-1
Content | Description | Ct value | dCT(NTC-Sample) | Melting curve matched to Pos control |
Sample | B6 (4/1) N6-No-Tre | 41.68 | -3.51 | y |
Sample | B7 (4/1) N6-No-Tre | N/A | N/A | n |
Sample | D2 (4/1) N6-No-Tre | 30.77 | 7.4 | n |
Sample | D4 (4/1) N6-No-Tre | 28.89 | 9.28 | n |
Sample | D5 (4/1) N6-No-Tre | 26.03 | 12.14 | n |
Sample | D7 (4/1) N6-No-Tre | 33.34 | 4.83 | y |
Sample | D8 (4/1) N6-No-Tre | 33.09 | 5.08 | y |
Sample | B3 (3/31) L4N9-Tre | 36.87 | 1.3 | n |
Sample | B4 (3/31) L4N9-Tre | 33.8 | 4.37 | y |
Sample | B6 (3/31) L4N9-Tre | N/A | N/A | y |
Sample | C2 (3/31) L4N9-Tre | 34.13 | 4.04 | n |
Sample | C5 (3/31) L4N9-Tre | 40.4 | -2.23 | y |
Sample | C7 (3/31) L4N9-Tre | 28.02 | 10.15 | n |
Sample | C8 (3/31) L4N9-Tre | 39.56 | -1.39 | y |
Sample | pos | 23.83 | ||
Blank | ntc | 38.17 | ||
Primer 4-1
Content | Description | Ct value | dCT(NTC-Sample) | Melting curve matched to Pos control |
Sample | B6 (4/1) N6-No-Tre | N/A | N/A | n |
Sample | B7 (4/1) N6-No-Tre | N/A | N/A | n |
Sample | D2 (4/1) N6-No-Tre | N/A | N/A | y |
Sample | D4 (4/1) N6-No-Tre | N/A | N/A | n |
Sample | D5 (4/1) N6-No-Tre | N/A | N/A | y |
Sample | D7 (4/1) N6-No-Tre | N/A | N/A | n |
Sample | D8 (4/1) N6-No-Tre | N/A | N/A | n |
Sample | B3 (3/31) L4N9-Tre | N/A | N/A | y |
Sample | B4 (3/31) L4N9-Tre | 38.75 | N/A | y |
Sample | B6 (3/31) L4N9-Tre | N/A | N/A | n |
Sample | C2 (3/31) L4N9-Tre | N/A | N/A | y |
Sample | C5 (3/31) L4N9-Tre | N/A | N/A | n |
Sample | C7 (3/31) L4N9-Tre | N/A | N/A | n |
Sample | C8 (3/31) L4N9-Tre | N/A | N/A | n |
Sample | pos | 23.51 | N/A | |
Blank | ntc | N/A | N/A | |
Primer 5-1
Content | Description | Ct value | dCT(NTC-Sample) | Melting curve matched to Pos control |
Sample | B6 (4/1) N6-No-Tre | 40.66 | -8.72 | n |
Sample | B7 (4/1) N6-No-Tre | 44.13 | -12.19 | n |
Sample | D2 (4/1) N6-No-Tre | 37.36 | -5.42 | n |
Sample | D4 (4/1) N6-No-Tre | 36.8 | -4.86 | n |
Sample | D5 (4/1) N6-No-Tre | 32.06 | -0.12 | y |
Sample | D7 (4/1) N6-No-Tre | 38.86 | -6.92 | n |
Sample | D8 (4/1) N6-No-Tre | N/A | N/A | n |
Sample | B3 (3/31) L4N9-Tre | 40.66 | -8.72 | n |
Sample | B4 (3/31) L4N9-Tre | N/A | N/A | y |
Sample | B6 (3/31) L4N9-Tre | N/A | N/A | y |
Sample | C2 (3/31) L4N9-Tre | 36.89 | -4.95 | n |
Sample | C5 (3/31) L4N9-Tre | N/A | N/A | y |
Sample | C7 (3/31) L4N9-Tre | 40.25 | -8.31 | n |
Sample | C8 (3/31) L4N9-Tre | N/A | N/A | n |
Sample | pos | 24.02 | ||
Blank | ntc | 31.94 | ||
Primer 6-1
Content | Description | Ct value | dCT(NTC-Sample) | Melting curve matched to Pos control |
Sample | B6 (4/1) N6-No-Tre | 38.26 | 0.89 | n |
Sample | B7 (4/1) N6-No-Tre | 34.57 | 4.58 | y |
Sample | D2 (4/1) N6-No-Tre | 42.1 | -2.95 | n |
Sample | D4 (4/1) N6-No-Tre | 39.49 | -0.34 | n |
Sample | D5 (4/1) N6-No-Tre | 33.99 | 5.16 | y |
Sample | D7 (4/1) N6-No-Tre | N/A | N/A | n |
Sample | D8 (4/1) N6-No-Tre | 28.96 | 10.19 | y |
Sample | B3 (3/31) L4N9-Tre | 34.64 | 4.51 | y |
Sample | B4 (3/31) L4N9-Tre | 39.92 | -0.77 | y |
Sample | B6 (3/31) L4N9-Tre | 35.97 | 3.18 | y |
Sample | C2 (3/31) L4N9-Tre | 36.08 | 3.07 | y |
Sample | C5 (3/31) L4N9-Tre | N/A | N/A | y |
Sample | C7 (3/31) L4N9-Tre | 33.26 | 5.89 | y |
Sample | C8 (3/31) L4N9-Tre | 37.54 | 1.61 | n |
Sample | pos | 26.85 | ||
Blank | ntc | 39.15 | ||